Amoebic gill disease and natriuretic peptide receptors in the gills of Atlantic salmon

Amoebic gill disease (AGD) is a problem in the farming of Atlantic salmon, and may compromise osmoregulatory, cardiovascular and respiratory functions. We examined the effects of AGD on atrial and C-type natriuretic peptide (ANP and CNP) stimulated branchial cyclic GMP formation, since natriuretic peptides (NPs) are involved in cardiovascular function and osmoregulation. NPs act via guanylyl cyclase receptors (NPR), which stimulate cGMP formation. NPR activity was measured by ANP and CNP stimulation of branchial cGMP formation, and compared between diseased and healthy salmon over an 11 day AGD infection. We also measured plasma osmolality. Osmolality increased in AGD infected salmon from an initial 355 mmol.kg-1 to 411 mmol.kg-1 at 11 days. There was no evidence that branchial cGMP formation changed in response to AGD. In all groups, CNP stimulation of guanylyl cyclase was 190% of basal rate, whereas ANP was 150% of basal. After 11 days, all groups were given a 4 h freshwater bath, the usual treatment for AGD. Another group was given a seawater to seawater transfer, to control for handling. In this group, plasma osmolality at 11 days was the same as in AGD fish. This elevation may be due to these fish experiencing disturbance for the first time in 11 days. ANP and CNP branchial NPR activity at the conclusion of the 4 h transfers was elevated in all groups compared to that at 11 days. The increased cGMP formation in the handling control suggests a NPR response to the transfer/handling stress. AGD fish demonstrated the greatest elevation in ANP and CNP guanylyl cyclase activity immediately following the bath; these values were greater than in the control groups. The AGD infected salmon, therefore, responded more emphatically to the freshwater treatment, suggesting that the NP system is involved in some aspects of AGD.

Brain neuropeptide Y and CCK and peripheral adipokine receptors : temporal response in obesity induced by palatable diet

Objective:
Palatable food disrupts normal appetite regulation, which may contribute to the etiology of obesity. Neuropeptide Y (NPY) and cholecystokinin play critical roles in the regulation of food intake and energy homeostasis, while adiponectin and carnitine palmitoyltransferase (CPT) are important for insulin sensitivity and fatty acid oxidation. This study examined the impact of short- and long-term consumption of palatable high-fat diet (HFD) on these critical metabolic regulators.

Methods:
Male C57BL/6 mice were exposed to laboratory chow (12% fat), or cafeteria-style palatable HFD (32% fat) for 2 or 10 weeks. Body weight and food intake were monitored throughout. Plasma leptin, hypothalamic NPY and cholecystokinin, and mRNA expression of leptin, adiponectin, their receptors and CPT-1, in fat and muscles were measured.

Results:
Caloric intake of the palatable HFD group was 2–3 times greater than control, resulting in a 37% higher body weight. Fat mass was already increased at 2 weeks; plasma leptin concentrations were 2.4 and 9 times higher than control at 2 and 10 weeks, respectively. Plasma adiponectin was increased at 10 weeks. Muscle adiponectin receptor 1 was increased at 2 weeks, while CPT-1 mRNA was markedly upregulated by HFD at both time points. Hypothalamic NPY and cholecystokinin content were significantly decreased at 10 weeks.

Conclusion:
Palatable HFD induced hyperphagia, fat accumulation, increased adiponectin, leptin and muscle fatty acid oxidation, and reduced hypothalamic NPY and cholecystokinin. Our data suggest that the adaptive changes in hypothalamic NPY and muscle fatty acid oxidation are insufficient to reverse the progress of obesity and metabolic consequences induced by a palatable HFD.

Ontogeny of putative GABAergic neurons and their receptors in the nervous system of the crayfish Cherax destructor

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

Natriuretic peptides and their receptors in the brain of the amphibian, Bufo marinus

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Role of leptin receptors in the development of obesity

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

Adrenergic regulation of carbohydrate metabolism during exercise

1. This series of studies was undertaken to examine the adrenergic regulation of carbohydrate metabolism during exercise. Recreationally active males were tested during moderate to intense exercise on a stationary cycle ergometer. Venous and arterial plasma obtained from indwelling catheters was analysed for hormonal and metabolite responses, and hepatic glucose production and glucose uptake were measured using the tracer-dilution method with stable isotopes. Muscle samples were obtained by the needle biopsy technique to examine muscle glycogen utilisation and the flux of related muscle metabolites using enzymatic, fluorometric and radioisotopic techniques. 2. During moderate exercise adrenaline infusion induced a marked hyperglycemia and this was due to reduced glucose uptake rather than enhanced hepatic glucose production. The reduction in glucose uptake was most likely mediated by a decrease in glucose phosphorylation, as indicated by the accumulation of glucose 6-phosphate with adrenaline infusion. 3. The hyperglycemic response to intense exercise was prevented by the administration of α- and β-adrenergic antagonists. Adrenergic blockade was without effect on hepatic glucose production whereas glucose uptake was enhanced when compared with control subjects. These data support the notion that adrenergic mechanisms are more important in restraining glucose uptake than enhancing hepatic glucose production during intense exercise. Other glucoregulatory factors are responsible for the increase in glucose production during intense exercise. 4. Elevated plasma adrenaline levels during moderate exercise in untrained men increases skeletal muscle glycogen breakdown and PDH activation which results

a7-Nicotinic acetylcholine receptors mediate an A71-42-induced increase in the level of acetylcholinesterase in primary cortical neurones

The β-amyloid protein (Aβ) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Aβ have suggested that this increase may be due to a direct action of Aβ on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Aβ increases levels of AChE in primary cortical neurones. Aβ₁₋₄₂ was more potent than Aβ₁₋₄₀ in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Aβ peptides. The effect of Aβ₁₋₄₂ on AChE was blocked by inhibitors of α7 nicotinic acetylcholine receptors (α7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of α7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Aβ₁₋₄₂ on AChE is due to an agonist effect of Aβ₁₋₄₂ on the α7 nAChR.

Irreversible and bivalent ligands for selected G-protein coupled receptors

Derivatives of pharmaceutical compounds that could potentially bind irreversibly to a hypertension controlling receptor were synthesised to provide tools for studying this receptor. Also compounds that simultaneously activate two cardiovascular receptors with opposing cellular mechanisms were synthesised. This resulted in a desired partial reduction in the activity of one receptor.

Identification, distribution and expression of natriuretic peptide receptors in trout

This study identified cell receptors for trout natriuretic peptide hormone. These hormones protect the heart and maintain fluid balance. Receptor populations on cells depend on whether the animal lives in freshwater or saltwater. Receptors are widely distributed and different receptor types perform different tasks. Their original evolutionary role is suggested.

Agonist allosteric enhancers at A1 adenosine receptors

Pharmaceutical agents that may be protective to the heart during ischemia were targeted in this project. Analogs to a lead compound containing potential cardioprotective properties were prepared. In the pharmacological evaluation five compounds were found to be more potent than the lead compound.